Summary
Macrophages
- Lining macrophage clusters were further assessed. The detection of
this population is inconsistent for the mock-infected replicates (higher
for r1, really low for r2, r3). This inconsistency is largely because
doublets are being disproportionately marked/filtered from the lining r2
and r3 populations. The data were reprocessed after reducing the
stringency of this step. This results in more consistent (and I think
accurate) detection of the lining population, and there is a more
reproducible reduction in this population in the CHIKV samples (which
supports previous data).
- Plots are included showing expression of the Culemann et al. gene
lists used for annotating the macrophage subsets.
- CHIKV+ cells were further assessed. Plots are included for the
previous approach where macrophages clusters are split into
CHIKV-high/low groups. The enrichment of CHIKV+ cells in specific
populations was also assessed using a hypergeometric test. The
populations with the highest fraction of CHIKV+ cells are lining and
infiltrating macrophages. However, the p-values are not impressive. I’m
going to see if I can come up with a better approach for presenting
these results.
- Plots are included summarizing differentially expressed genes for
mock vs CHIKV for all macrophages grouped together, and for each
individual subset.
- There are two key background signals that are observable when
looking at genes downregulated in the CHIKV samples.
- The mock samples are generally lower quality and have a higher
proportion of mitochondrial counts, to account for this, mitochondrial
genes have been omitted from the figures.
- The mock samples also show higher expression of genes associated
with skeletal muscle in all cell types (troponin, myosin genes). In 28
dpi datasets there are two clusters annotated as skeletal muscle,
however, these cells are generally too large for the microfluidics used
in the 10x platform. Many of these muscle cells are likely not fully
intact and the mRNA originating from these cells likely contaminated the
buffer during capture. There is no reason to believe this background
signal is skewing our results, but this explains why troponin/myosin
genes are differentially expressed (for publication it would be
reasonable to omit these genes and explain why).
- Plots are included summarizing expression of MHC class II genes for
mock vs CHIKV for each subset.
- Plots were regenerated for the Simmons et al. gene lists after
adjusting QC filtering as described above.
T cells
- Plots are included for marker genes for different T cell
subsets.
- Plots are included showing Ifng expression for all subsets mock vs
CHIKV.
- Plots are included summarizing differentially expressed genes for
mock vs CHIKV for all T cells grouped together, and for CD4/CD8 T cells
grouped together.
UMAP projections show sample groups (left), cell type annotations
(middle), and CHIKV+ cells (right).

The fraction of cells passing QC filtering cutoffs is shown for each
sample.

Macrophages
Subset annotations
UMAP projections show macrophage subsets.

Module scores for Culemann et al. gene lists are shown for each
macrophage cluster. Clusters are colored based on the assigned subset
labels.

Heatmaps show mean expression of Culemann et al. gene lists.
Replicates are shown separately for each macrophage subset.
CCR2_ARG1_infiltrating

CCR2_IL1B_infiltrating

CX3CR1_lining

RELMa_interstitial

AQP1_interstitial

CHIKV+ cells
UMAP projections show CHIKV+ cells (left) and CHIKV-high clusters
(middle, right) for CHIKV-infected samples.

The fraction of cells that are CHIKV+ is shown for each replicate
from each macrophage subset. P values were calculated by pooling
replicates together, using a hypergeometric test with Bonferroni
correction.

The fraction of CHIKV+ cells passing QC filtering cutoffs is shown
for each sample.

Differential expression
Top 50 upregulated genes associated with arthritis are shown below
for mock- vs CHIKV-infected macrophages (all subsets grouped
together).
Genes were filtered for those where the change in expression was in
the same direction (up or down) for all replicates, an adjusted p value
< 0.05 and an absolute log2 fold change > 0.25 for at least 2/3
replicates

Genes associated with antigen presentation via MHC class
II are shown below. Boxplots compare expression in mock (light)
vs CHIKV (dark) for each macrophage subset.
